(1) Field of the Invention
The present invention relates to an assay for assessing the activity of CYP2C9 and the potential of an analyte to modulate CYP2C9 activity, e.g., inhibitor or inducer of CYP2C9 activity. The assay determines CYP2C9 activity by measuring 4′-hydroxylation of diclofenac in reactions comprising CYP2C9, microsomes comprising CYP2C9, or hepatocytes using diclofenac labeled with tritium in the 4′ position as a substrate and a sorbent which preferentially binds non-polar compounds such as diclofenac to separate the labeled diclofenac from tritiated water formed during hydroxylation of the labeled diclofenac at the 4′ position by CYP2C9. The assay is useful for assessing CYP2C9 enzymatic activity and CYP2C9 inhibition or induction potential of drug candidates in order to exclude potent CYP inhibitors or inducers from further development.
(2) Description of Related Art
The pharmacokinetic and toxicokinetic properties of pharmaceuticals depend in great part on their biotransformation by drug metabolizing enzymes. The main drug metabolizing system in mammals is cytochrome P450 (CYP), a family of microsomal enzymes present predominantly in the liver. Multiple isoforms of CYP catalyze the oxidation of chemicals of endogenous and exogenous origin, including drugs, steroids, prostanoids, eicosanoids, fatty acids, and environmental toxins (Ioannides, In Cytochromes P450. Metabolic and Toxicological Aspects. CRC Press, Boca Raton. (1996)). If a drug that is metabolized by a particular CYP isozyme is co-administered with an inhibitor of that same enzyme, changes in its pharmacokinetics can occur, which can give rise to adverse effect (Bertz and Granneman, Clin. Pharmacokinet. 32: 210-258 (1997); Lin and Lu, Clin. Pharmacokinet. 35: 361-390 (1998); Thummel and Wilkinson, Ann. Rev. Pharmacol. Toxicol. 38: 389-430 (1998); von Moltke et al., Biochem. Pharmacol. 55: 113-122 (1998)). It is therefore important to be able to predict and to prevent the occurrence of clearance changes due to metabolic inhibition. During the drug discovery process, it is routine practice in the pharmaceutical industry to assess CYP inhibition potential of drug candidates in order to exclude potent inhibitors from further development (Lin and Lu, ibid. (1998); Crespi and Stresser, J. Pharmacol. Toxicol. Methods 44: 325-331 (2000); Bachmann and Ghosh, Curr. Drug Metab. 2: 299-314 (2001); Riley, Curr. Opin. Drug Disc. Dev. 4: 45-54 (2001)).
The polymorphically expressed CYP2C9 is one of the most important drug metabolizing enzymes in humans. It constitutes about 20% of the total human liver CYP content and metabolizes about 10% of therapeutically important drugs (Miners and Birkett, Br. J. Pharmacol. 45: 525-538 (1998); Goldstein, Br. J. Clin. Pharmacol. 52: 349-355 (2001); Xie et al., Adv. Drug Deliv. Rev. 54: 1257-1270 (2002); Schwarz, Eur. J. Clin. Invest. 33: 23-30 (2003)). Many clinically relevant drug interactions due to inhibition of CYP2C9 have been described (Miners and Birkett, ibid.; Ito et al., Br. J. Clin. Pharmacol. 57: 473-486 (2004)). Several assay methods are currently used for determining the potential of drug candidates to inhibit CYP2C9 activity, and each of these methods presents distinct advantages and disadvantages. The most widely used marker reactions are diclofenac 4′-hydroxylation, tolbutamide 4′-hydroxylation, and S-warfarin 7′-hydroxylation. Inhibition assays are typically conducted using human liver microsomes (HLM) as the enzyme source and high pressure liquid chromatography (HPLC) with ultraviolet (UV) or mass spectrometric (MS) detection for quantifying the hydroxylated substrate.
Because of the need to use HPLC to isolate and detect the hydroxylated substrate, current assays for detecting inhibitors of CYP2C9 are not suited for high throughput screening. In an attempt to create an assay that is suitable for high throughput screening, a number of fluorogenic substrate probes have been developed. These probes enable detection of the hydroxylated substrate by measuring fluorescence. There is no need to separate the product from the other components of the assay. The ability to detect the product without having to isolate the product by HPLC provides an assay practical for use in a high throughput screening format. However, using fluorogenic probes have several disadvantages. First, the fluorogenic probes are frequently metabolized by more than one CYP isoform; therefore, the assays have to be conducted using a single CYP isoform (produced by recombinant DNA technology) instead of HLM. Second, the results of assays that use the fluorogenic probes and a recombinant CYP isoform do not correlate well with results obtained using conventional probes in HLM (Cohen et al. Drug Metab. Dispos. 31, 1005 (2003)). The reason for the lack of correlation may include metabolism of test inhibitors by enzymes present in the HLM but not in assays that use a recombinant CYP isoform and the existence of multiple substrate binding sites.
Therefore, there remains a need for an assay for identifying CYP2C9 modulators that is based on using diclofenac as the substrate, is at least as sensitive and specific as the conventional assays, and is readily adaptable to a high throughput screening format. There is also a need for an assay for assessing CYP2C9 activity in hepatocytes.